Paraffin Embedding

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Guy Sovak
Guy Sovak's picture
Paraffin Embedding

Hi all,
I have a muscle tissue which I want to embed in Paraffin.
I am going to do it like follows:
1- Over night in melted paraffin
2- 1Hr. Melted paraffin
3- 1 Hr. Melted paraffin.

The question is what temp. the paraffin should be , 58deg Cel is the melting temp.
Some protocol say to do it in 60 other say 65 deg Cel?

Any experience with muscle tissue paraffin embedding?
Guy

Kvachhani
Kvachhani's picture
yes 60 degree C is fine, i

yes 60 degree C is fine, i had done lots of time with this and it works perfectly. higher than this temp. may cause hardness of tissue and cause dust formation during sectioning.
regards

Guy Sovak
Guy Sovak's picture
Thanks for your response,

Thanks for your response,
I had dust formation couple of times and it is also could arise from wrong dehydration.
I have another question to you,
When I am cutting the muscle tissue many times I some how lose the connecting tissue. So when staining it in Masson Trichrome I can see just the muscle bundles and some times the neuro vascular bundle. Do you have any ideas why I am getting this?
 

Kvachhani
Kvachhani's picture
yes improper dehydration may

yes improper dehydration may cause dust formation during sectioning.
Regarding loss of CT during sectioning, this may cause due to improper paraffine embeding. You may do one thing that after processing of tissue, keep it in melted paraffine before making block (embeding), when you starts block making remove tissue from melted parraffine and make block immideatly which enhance the proper block making and make shoure that blade / knife is sharpe enough during section cutting. this may solve your problem.
regards.

Arvind Singh Pundir
Arvind Singh Pundir's picture
guy wrote:Hi all, I have a

guy wrote:

Hi all, I have a muscle tissue which I want to embed in Paraffin. I am going to do it like follows: 1- Over night in melted paraffin 2- 1Hr. Melted paraffin 3- 1 Hr. Melted paraffin. The question is what temp. the paraffin should be , 58deg Cel is the melting temp. Some protocol say to do it in 60 other say 65 deg Cel? Any experience with muscle tissue paraffin embedding? Guy

 
as far as i think its not good to keep the tissue overnight at melting point as it can lead to protein destruction , and the temp should be 2 to 4 degree more than the MP of the wax you are using

Kvachhani
Kvachhani's picture
yes overnight keeping in

yes overnight keeping in paraffine is not necessory. but the temperature of the paraffine should around 60 degree C, as their are product of praffine which have higher malting point and if you keep tissue in that may cause protein denaturation.
regards.

Guy Sovak
Guy Sovak's picture
Thanks all,

Thanks all,
I have lowred the temp. to 59deg cel.
I will do the paraffinisation 3 times 60 min each.
Another Question?
After dehydration last step  Xylene, can I leave it over night at RT out side the Xylene on the bench before putting it in the melted paraffin?
Guy

Arvind Singh Pundir
Arvind Singh Pundir's picture
 after dehydration you have

 after dehydration you have to put it in xylene and later on in parraffin , keeping it in xylene overnight will make your tissue brittle

Kvachhani
Kvachhani's picture
guy wrote:

guy wrote:

Thanks all,
I have lowred the temp. to 59deg cel.
I will do the paraffinisation 3 times 60 min each.
Another Question?
After dehydration last step  Xylene, can I leave it over night at RT out side the Xylene on the bench before putting it in the melted paraffin?
Guy

It is not desirable to keep tissue out side, rather you can keep tissue in melted parraffine after xyline stape and let that parraffine solidify, in this you can keep it over night and very next day you can again start the process from parraffine - I, II, & III.
So stapes are
Xyline
keep tissue in melted Paraffine
Allow to solidify and keep it in for over night.
Next day start process - melted parraffine I, II, & III.
Regards.

excalibur
excalibur's picture
When processing any tissue

When processing any tissue the temp of the paraffin should not exceed 60C.
It sounds like you are processing by hand and not on an automated processor.
First. Fixation. Fixation. Fixation. How big is your specimen? Unless larger than 1cm cubed, overnight in PFA or formalin should be fine.
Proceesing should be 70% EtOH , 2 changes 95% EtOH, 3 changes 100% EtOH, 2-3 changes xylene  30-45 mins each
3 paraffins 45mins to 1 hour.
If you cannot process in one day, the tissue should be left in the 70 or 95% EtOH. NEVER, ever leave the tissue out of a solution to dry!!!!!!!!!!!!!
 

Guy Sovak
Guy Sovak's picture
Thanks Excalibur,

Thanks Excalibur,
The sections are 0.5X0.5X0.5mm.
10% NBF for 5 days ( I know that 24 hr is OK)
The exchanges are done in 70% then 80% then 95% ( I tried each for 30 up to 60min)
100% I have used 95% Ethol + 5% meth, or 95% Ethol + 5% Isopro. 3 changes 1hr each
Xylene 2 Changes 30min up to 60min.
Paraffin below 60deg cel 3 changes two ways either first change Over night and then the other 2 1hr each, or all the three changes all together 2hr (40min each).
After paraffin I embeded the tissue.
Stii none of the above resulted in good sectioning. The tissue crumbles turns to dust.
I do not have any Idea what to do?
Thanks for helping all of you.
This is what Scientist Solutions is all about , people from different contintents helpping each other.
Guy
 

excalibur
excalibur's picture
If you are using 95% EtOH and

If you are using 95% EtOH and then adding 5% MeOH or Iso, the 95% still contained 5% water, which is probably your problem. There should be no water content at all in your 100% alcohol to process.
Are you using a 95% alcohol (5% water) or reagent alcohol which contains 95% EtOH and 5% other alcohols and no water?
Is the xylene turning a cloudy white when you place the tissue in it from the alcohol? If it is, there is water in your alcohol.
 

Platelet Pete
Platelet Pete's picture
Hi. Platelet Pete here. I

Hi. Platelet Pete here. I have been having some of the same issues with crumbling of tissues. In my case, intestine, lung and brain are okay with my processing method. I have problems with kidney, liver and spleen. I thought the issue was the tissue composition, that highly vascularized. This happened regardless if I used fresh alchols and xylene subsititute or xylene. We are using the same protocol as our histology core and they don't appear to have this problem. I thought it might be the xylene subsitute but didn't consider the paraffin temperature. I hope this helps.
I will subscribe to this forum.

Arvind Singh Pundir
Arvind Singh Pundir's picture
guy wrote:

guy wrote:

Thanks Excalibur,
The sections are 0.5X0.5X0.5mm.
10% NBF for 5 days ( I know that 24 hr is OK)
The exchanges are done in 70% then 80% then 95% ( I tried each for 30 up to 60min)
100% I have used 95% Ethol + 5% meth, or 95% Ethol + 5% Isopro. 3 changes 1hr each
Xylene 2 Changes 30min up to 60min.
Paraffin below 60deg cel 3 changes two ways either first change Over night and then the other 2 1hr each, or all the three changes all together 2hr (40min each).
After paraffin I embeded the tissue.
Stii none of the above resulted in good sectioning. The tissue crumbles turns to dust.
I do not have any Idea what to do?
Thanks for helping all of you.
This is what Scientist Solutions is all about , people from different contintents helpping each other.
Guy
 

Dear Sir
as i am reading this discussion one thing is sure that your tissue was left in 10%NBF for 5 days thats a too much of fixation, as you have not mentioned the thickness of your tissue before fixation, other thing is there are chances of removal of too much of bound water being released from the tissue  during dehydration as the dehdration has to remove only the free water molecules , the possible remedy for this may be keep your block before sectioning on ice or at 4 degree and keep moistening the exposed part of the tissue during cutting as it may compensate a bit for your excess dehdration if it had happened, rest is all irreversible you have try at new tissue with fresh protocol keeping the points raised in discussion at handy.....we will continue the discussion as it will cetainly expose the culprit...............................

excalibur
excalibur's picture
You cannot overfix. Once the

You cannot overfix. Once the crosslinks are formed, the tissue can not undergo any more changes. IMHO, good fixation is the most important step.
As to your tissue crumbling during sectioning, this is caused by improper processing. The most likely culprit is water in the last alcohols. I have 30 years experience in histology and this is my opinion without actually seeing your specimens.

Guest (not verified)
Guest's picture
Hi All,

Hi All,
I would like to add a couple of comments:
1) Muscle is usually not pariffin embedded, it is cut fresh.  Muscle physiologists and pathologists usually use fresh frozen muscle (not fixed) to look at ATPase's, conective tissue, blood vessels ect.  You can also do maison's ect.  If you are going to do any of the normal stains on muscle then I respectfully disagree with the previous posts about fixation time.  Normally you place the frozen section on a gelatin coated slide, ary dry and then fix in PFA for 5 sec, rinse and then stain.  Any longer fixation will destroy the ezymatic staining.
2) fixation and immunohistochemistry,   Different antigens respond differently to fixation time.  I aggree that some antigens can be left in fixative for days- weeks  without interfering with the immunohistochemisty.  Others we use very limited amounts.  For sensitive antigens we use cryosectionig: Mice are perfused with 30 ml and then the tissue  placed in PBS at 4oC for one hour, the solution replaced PBS with 30 % sucrose and then stored for cutting at 4 c for up to a week.  
 Some antigens are unmasked by parifin embedding and others are destroyed by the heating to 60 degrees.

excalibur
excalibur's picture
Muscle tissue for enzymatic

Muscle tissue for enzymatic staining should be from fresh, UNFIXED, snap frozen sections.
Short fixation times for frozen sections is essentially no fixation and defeats the whole purpose of doing a frozen. Why fix at all?
maisons = Masson's trichrome?
 

Guest (not verified)
Guest's picture
I agree muscle should be snap

I agree muscle should be snap frozen  UNFIXED  at its natural length.
When you re-hydrate the muscle it tends to contract (it is fantastic to watch) and comes it off the slide. The only reason to have a very short dip in fix is to stick the muscle to the gelatin on the slide.
 
 

Guy Sovak
Guy Sovak's picture
Hi again,

Hi again,
I had the same problem with tissues that were fxed for 24 and 48Hr.
The sample is 1cm thick.
Next step woould be to cut it into small cubes. 0.5X0.5X0.5cm
Thanks
Guy

excalibur
excalibur's picture
Hello guy,

Hello guy,
I still think the problem is water in your alcohols. Water and xylene do not mix!
You are not getting true dehydration for the xylene to clear the tissue and thus the paraffin is not infiltrating.
It is a Process. Alcohol to remove the water. Xylene to remove the alcohol. Paraffin to replace the xylene.
 

Arvind Singh Pundir
Arvind Singh Pundir's picture
Hi guy

Hi guy
i think you should check all the reagents whether they are pure, or else i have a resource by which you can make out whether the alcohol you are using is absolute or not as it seems water is the main culprit behind the prob.
 "How can I determine whether used "absolute" alcohol is still OK for the last stage of dehydrating specimens or slides ? " www.biologicalstaincommission.org/index.html
 

peter36
peter36's picture
improper dehydration leads to

improper dehydration leads to improper clearing and improper wax penetration. Tissue will appear soft and tend to explode on the water bath. Crunchy tissue is usually caused by extended xylene times on H@E staining the edges will appear very pink with eosin staining "cooked"

sumitnbrc
sumitnbrc's picture
you can follows

you can follows
           1 -24 h in melted paraffin 58deg cel.
           2-   30min in  melted paraffin  60 deg cel.
           3-    15 min in melted paraffin 60  deg cel .

Myofib
Myofib's picture
My student is having this

My student is having this problem today, soft tissue (human skin) that explodes in the water bath. We are going to melt and return it to 95% o/n then finish the dehydration slowly. It is being done by hand. I have done this a number of previous times without any trouble. I think the xylenes are contaminated.

efrainmax
efrainmax's picture
tissue explosion on the water

tissue explosion on the water bath sometimes is caused by high temp on the water bath (just an observation :))

Kaja K
Kaja  K's picture
paraffin embedding for 300um brain slices

Hello,in our lab we established paraffin embedding in thicker parts of organs yet we have troubles with freshly cut 300um rat brain slices.Firstly, the slices get wrinkled. Therefore I cover them during the fixation (24h) and uncover them during dehydration until xylene. I can see they are getting wrinkled during the hydration yet I don't want them to cover with anything because the process might not be efficient then.  At that point (during xylene clearing and wax infiltration) I keep them in cassettes filled with sponges. But in the end, they are still a bit wrinkled. I can see it when cutting the slices, there are holes in my 10um slices. Since I started to use the sponges it got better but still not enough.Secondly, the slices get fragile and brittle almost immediately when I put them in xylene. The xylene appears transparent so I don't think the water is the problem.Thirdly, when I embed the slices, I put the slices in a hot wax and transfer the mould on a cold surface so that the wax hardens very quickly. Has anyone tried with pressing the slices against the mould?The protocol is fixation overnight, 3x PBS wash, 2x 30min 50% etOH, 70% etOH overnight, 95% etOH/ 5% methanol 1h, 100% etOH 3x30min, 50% etOH/50% xylene 30min, xylene 2x 1h, first wax 1h, second wax overnight, embedding. The fixation lasts for 24h but I shortened the rest of the protocol by half and I don't see any difference. At least in these thin slices. Hope anyone has some experience in this,best wishes,K